Our section has a major effort devoted to understanding the molecular and cellular basis of stuttering, a common but poorly understood speech disorder. Stuttering has long been known to have a genetic component, and we have previously identified mutations in the GNPTAB, GNPTG and NAGPA genes that are associated with stuttering in populations worldwide. These results indicate that non-syndromic stuttering can be associated with partial loss-of-finction mutations in lysosomal targeting and transport, a cellular function that is well-studied in health and disease. More recently we have identified mutations in the AP4E1 gene that cause stuttering. AP4E1 encodes a cellular component that serves to direct movement of vesicles within the cell, including those destined to go to the lysosome. The product of the AP4E1 genes recognizes and directs the trafficking of the product of the NAGPA gene in particular, thus tying this finding to our previous gene findings, and highlighting the importance of this cellular process in the genesis of stuttering. During the past year we have focused on research to determine how the cell metabolic defects caused by mutations in these genes lead to stuttering without any other discernible symptoms. To facilitate these and other studies, we have developed a mouse model of human stuttering. This was done by creating so-called knock-in strains of mice that carry the mutations identified in humans who stutter. These experiments require a detailed acoustical analysis of mouse vocalization, which is largely ultrasonic in nature. In these experiments, we are working with Drs. Terra Barnes and Tim Holy at Washington University in St. Louis, and by Dr. Tae-Un Han in our Section. Our studies have demonstrated that the presence of human stuttering mutations causes reproducible alterations in mouse vocalizations, and that these alterations show parallels with the alterations present in the speech of the individuals who stutter and carry such mutations. In the past year, we used these mice to identify specific anatomic and cellular alterations that accompany this vocalization deficit using three different approaches. The first approach was staining brain tissue with antibodies that recognize surface molecules on specific cell types in the brain, and comparing these cells in mutant animals with those in their wild-type littermates. The second approach used additional mouse genome engineering techniques to express stuttering mutations within specific neuronal cell types and cell lineages within the brain using neuronal cre- driver lines. Testing the ultrasonic vocalizations of these mice are expected to provide an independent but parallel approach to identify specifically which cell types within the brain mediate these alterations in vocalization. The third approach looked at larger scale brain structures in these mice using MRI and DTI, done in collaboration with the NIH Mouse Imagining Facility using a 14 Tesla scanner. These mouse studies resulted in a surprising finding of highly specific deficits in astrocytes, a glial cell type within the brain, and that these deficits were concentrated in the corpus callous, the major white matter tract that connects the right and left hemispheres of the brain. Subtle but significant differences in these white matter tracts were also observed by DTI scanning in these mice. These findings support the hypothesis that stuttering results from a failure of coordination of activities across the two sides of the brain, and were reported in a prominent paper by Han et al, published in the Proceedings of the National Academy of Sciences, USA. Our studies of taste perception are focused on the role of genetic differences in taste perception in tobacco use, particularly the use of mentholated cigarettes, which are disproportionately used by African Americans. Our goal is to determine whether this disproportionate use is associated with genetic differences, specific to African Americans, in genes that encode taste perception machinery. This study is being done in collaboration with the University of Texas Southwestern Medical Center, using the well-characterized Dallas Heart Study population. In the past year, we have also entered into a collaboration with Dr. Thomas Kirchner at New York University and Dr. Ray Niaura at the Schroeder Center for Tobacco Research, who together have provided >1700 DNA samples from individuals with known smoking phenotypes. In the past year, whole exome genotyping and sequencing have identified a surprising association with a coding variant in the MRGPRX4 gene. This variant exists only in populations of African origins. While the function of MRGPRX4 is poorly understood, it is expressed primarily in sensory neurons, where it appears to be involved in nociception. These findings were published in a prominent paper by Kozlitina et al in PLoS Genetics..